Restraint Stress-Induced Immunosuppression Is Associated with Concurrent Macrophage Pyroptosis Cell Death in Mice

Psychological stress is widely acknowledged as a major contributor to immunosuppression, rendering individuals more susceptible to various diseases. The complex interplay between the nervous, endocrine, and immune systems underlies stress-induced immunosuppression. However, the underlying mechanisms of psychological-stress-induced immunosuppression remain unclear. In this study, we utilized a restraint stress mouse model known for its suitability in investigating physiological regulations during psychological stress. Comparing it with cold exposure, we observed markedly elevated levels of stress hormones corticosterone and cortisol in the plasma of mice subjected to restraint stress. Furthermore, restraint-stress-induced immunosuppression differed from the intravenous immunoglobulin-like immunosuppression observed in cold exposure, with restraint stress leading to increased macrophage cell death in the spleen. Suppression of pyroptosis through treatments of inflammasome inhibitors markedly ameliorated restraint-stress-induced spleen infiltration and pyroptosis cell death of macrophages in mice. These findings suggest that the macrophage pyroptosis associated with restraint stress may contribute to its immunosuppressive effects. These insights have implications for the development of treatments targeting stress-induced immunosuppression, emphasizing the need for further investigation into the underlying mechanisms.


Introduction
The effects of psychological stress on the immune system and its ability to trigger immunosuppression have been extensively examined [1][2][3][4][5].It is well-established that chronic or prolonged psychological stress can lead to dysregulation of immune function, resulting in increased susceptibility to infections, delayed wound healing, and heightened risk of autoimmune and inflammatory diseases.The intricate interplay between the nervous, endocrine, and immune systems is involved in the mechanisms of psychologicalstress-induced immunosuppression.However, the specific details of these mechanisms remain elusive.
The restraint stress model is a well-recognized tool to study psychological-stressrelated physiological, behavioral, and biochemical changes in mice [6][7][8][9].It has been shown that chronic restraint stress can lead to immunosuppression [10][11][12][13].However, the impact of psychological alterations linked to acute restraint stress on the induction of fed restraint stress mouse model, we conducted the open field test (OFT) mouse behavior test according to previously reported methods [49,50].The results revealed that mice subjected to Evans-blue-fed restraint stress treatment exhibited anxiety-related behavior, providing evidence of psychological stress's contribution to this model (Figure 1).These findings are consistent with numerous previous reports that have identified psychological stress as one of the major factors associated with restraint stress animal models [6][7][8]16,[51][52][53][54][55][56][57][58][59][60][61][62].Additionally, as psychological-stress-induced immunosuppression is rarely discussed, this model is suitable to address this question.

Restraint Stress Resulted in the Development of Anxiety-like Behaviors in C57Bl/6J Mice
Restraint stress exposes experimental mice to both physiological and psychological stresses.To investigate the potential involvement of psychological stress in the Evans-blue-fed restraint stress mouse model, we conducted the open field test (OFT) mouse behavior test according to previously reported methods [49,50].The results revealed that mice subjected to Evans-blue-fed restraint stress treatment exhibited anxiety-related behavior, providing evidence of psychological stress's contribution to this model (Figure 1).These findings are consistent with numerous previous reports that have identified psychological stress as one of the major factors associated with restraint stress animal models [6][7][8]16,[51][52][53][54][55][56][57][58][59][60][61][62].Additionally, as psychological-stress-induced immunosuppression is rarely discussed, this model is suitable to address this question.During the 20 h stress procedure, both the no stress and stress groups were deprived of access to food and water.After the termination of restraint stress, both the unstressed and stressed groups of mice were given access to food and water for a period of 2 h to restore their resources.Subsequently, the OFT was conducted.Representative video tracking images captured during a 5-min OFT are presented (A).A comparison was made between the control group (no stress) and the restraint stress group (stress) for total distance traveled (B), distance traveled in the central zone (C), and time spent in the central area (D).The number of samples used for analysis was 3 (n = 3).* indicates statistical significance at p < 0.05, ** indicates statistical significance at p < 0.01, when compared to their respective no stress groups.

The Mechanism of Immunosuppression Differs between Restraint Stress, Intravenous Immunoglobulin (IVIg), and Ambient Cold Exposure in Mice
The immunosuppression property of IVIg is evident from its ability to ameliorate ITP [19,31,63,64].We have previously shown that ambient cold exposure inducedimmunosuppression through an IVIg-like pathway in mice, which could be typically induced in wild-type but not in P-selectin deficient (Selp −/− ) mutant mice [18].Using amelioration on ITP as previously described [18,19], the restraint-stress-induced immunosuppression was demonstrated in mice (Figure 2).Here we found that treatments of IVIg, cold exposure, and restraint stress all ameliorated anti-CD41 Ig injection-induced ITP in wild-type mice (Figure 2A,D,G experiment outline; Figure 2B,E,H).This indicated that restraint stress exerts an immunosuppressive effect as it successfully reduced autoantibody-induced thrombocytopenia (Figure 2H).In agreement with our previous report, such rescue on the ITP cannot be observed in Selp −/− mutant mice (Figure 2C,F).Intriguingly, here we found that restraint stress can ameliorate ITP in Selp −/− mice (Figure 2I).Because IVIg and cold exposure exert P-selectin-dependent rescue on ITP, these results suggest that restraint stress exerts immunosuppression through a different pathway, which is independent of P-selectin.After the termination of restraint stress, both the unstressed and stressed groups of mice were given access to food and water for a period of 2 h to restore their resources.Subsequently, the OFT was conducted.Representative video tracking images captured during a 5-min OFT are presented (A).A comparison was made between the control group (no stress) and the restraint stress group (stress) for total distance traveled (B), distance traveled in the central zone (C), and time spent in the central area (D).The number of samples used for analysis was 3 (n = 3).* indicates statistical significance at p < 0.05, ** indicates statistical significance at p < 0.01, when compared to their respective no stress groups.

The Mechanism of Immunosuppression Differs between Restraint Stress, Intravenous Immunoglobulin (IVIg), and Ambient Cold Exposure in Mice
The immunosuppression property of IVIg is evident from its ability to ameliorate ITP [19,31,63,64].We have previously shown that ambient cold exposure inducedimmunosuppression through an IVIg-like pathway in mice, which could be typically induced in wild-type but not in P-selectin deficient (Selp −/− ) mutant mice [18].Using amelioration on ITP as previously described [18,19], the restraint-stress-induced immunosuppression was demonstrated in mice (Figure 2).Here we found that treatments of IVIg, cold exposure, and restraint stress all ameliorated anti-CD41 Ig injection-induced ITP in wild-type mice (Figure 2A,D,G experiment outline; Figure 2B,E,H).This indicated that restraint stress exerts an immunosuppressive effect as it successfully reduced autoantibody-induced thrombocytopenia (Figure 2H).In agreement with our previous report, such rescue on the ITP cannot be observed in Selp −/− mutant mice (Figure 2C,F).Intriguingly, here we found that restraint stress can ameliorate ITP in Selp −/− mice (Figure 2I).Because IVIg and cold exposure exert P-selectin-dependent rescue on ITP, these results suggest that restraint stress exerts immunosuppression through a different pathway, which is independent of P-selectin.Evidence has consistently shown that macrophages play a critical role in the phagocytosis and clearance of autoantibody-bound and opsonized platelets in ITP [65][66][67].Additionally, macrophages are known to play a crucial role in clearing invading bacteria during infections [68][69][70].Based on these findings, we conducted an analysis of bacterial clearance as described [18], to investigate whether the observed immunosuppressive effects also affect macrophage function (Figure 3).Consistent with the results presented in Figure 2, we discovered that treatments involving IVIg, cold exposure, and restraint stress all suppressed bacterial clearance in wild-type mice (Figure 3A,C,E experimental outline; Figure 3B,D,F WT groups), indicating their immunosuppressive properties.However, such suppression of bacterial clearance was not observed in Selp −/− mutant mice following IVIg and cold exposure treatments (Figure 3B,D), but it was evident in Selp −/− mice after 9 h of restraint stress (Figure 3F).These findings strongly suggest that restraint stress induces immunosuppression through a distinct pathway that operates independently of P-selectin.Evidence has consistently shown that macrophages play a critical role in the phagocytosis and clearance of autoantibody-bound and opsonized platelets in ITP [65][66][67].Additionally, macrophages are known to play a crucial role in clearing invading bacteria during infections [68][69][70].Based on these findings, we conducted an analysis of bacterial clearance as described [18], to investigate whether the observed immunosuppressive effects also affect macrophage function (Figure 3).Consistent with the results presented in Figure 2, we discovered that treatments involving IVIg, cold exposure, and restraint stress all suppressed bacterial clearance in wild-type mice (Figure 3A,C,E experimental outline; Figure 3B,D,F WT groups), indicating their immunosuppressive properties.However, such suppression of bacterial clearance was not observed in Selp −/− mutant mice following IVIg and cold exposure treatments (Figure 3B,D), but it was evident in Selp −/− mice after 9 h of restraint stress (Figure 3F).These findings strongly suggest that restraint stress induces immunosuppression through a distinct pathway that operates independently of P-selectin.Here we utilized a previously established mouse model that involved administe ing Evan blue dye to measure GI leakage levels at various time courses during restrai stress [14][15][16][17].By employing this Evans-blue-fed mouse model, we made several not worthy observations.For example, we found a clear kinetic association between r straint-stress-induced GI leakage and increased infiltration of macrophages into th spleen, indicating a potential link between these two phenomena (Figure 4B vs. Figu 4C).
During the restraint stress experiment, we observed the peak levels of all measure parameters at the 9 h mark.Increased GI leakage, splenic macrophage infiltration, an elevated stress hormone levels (corticosterone and cortisol) were most pronounced this time point, indicating a time-dependent response to restraint stress (Figu 4B,C,E,F).
Furthermore, the infiltration of macrophages into the spleen strongly implies the involvement in the aberrant physiological effects induced by restraint stress.The fa that macrophage infiltration was detected within the spleen, a key organ of the immun system, further supports their potential role in mediating the observed abnormaliti triggered by restraint stress (Figure 4C).Here we utilized a previously established mouse model that involved administering Evan blue dye to measure GI leakage levels at various time courses during restraint stress [14][15][16][17].By employing this Evans-blue-fed mouse model, we made several noteworthy observations.For example, we found a clear kinetic association between restraint-stressinduced GI leakage and increased infiltration of macrophages into the spleen, indicating a potential link between these two phenomena (Figure 4B vs. Figure 4C).
During the restraint stress experiment, we observed the peak levels of all measured parameters at the 9 h mark.Increased GI leakage, splenic macrophage infiltration, and elevated stress hormone levels (corticosterone and cortisol) were most pronounced at this time point, indicating a time-dependent response to restraint stress (Figure 4B,C,E,F).
Furthermore, the infiltration of macrophages into the spleen strongly implies their involvement in the aberrant physiological effects induced by restraint stress.The fact that macrophage infiltration was detected within the spleen, a key organ of the immune system, further supports their potential role in mediating the observed abnormalities triggered by restraint stress (Figure 4C).

Restraint-Stress-Induced Immunosuppression Is Associated with Reduced Phagocyte Function and Increased Cell Death in Mice
Upon observing relatively high levels of viable bacteria several hours after bacterial injection (Figure 3F) and higher spleen infiltration of macrophages (Figure 4C), we postulated that the impaired phagocyte function of splenic macrophages, specifically their ability to engage and engulf bacteria, might be responsible for restraint-stress-induced immunosuppression.Using described methods [19,71] to assess the phagocyte function of splenic macrophages, we employed fluorescent nanoparticles (NPs) as phagocyte targets.Intriguingly, we discovered that treatments involving IVIg, cold exposure, and restraint stress all resulted in a suppression of spleen phagocyte engagement with fluorescent NPs (Figure 5).Subsequently, to explore the suppressive effect on the live phagocyte population, we examined NP engagements with live splenocytes and observed that restraint stress also suppressed the NP-engaging ability of live splenocytes (Figure S1).These findings strongly suggest that the suppression of phagocyte function contributes to the immunosuppressive effects induced by IVIg, cold exposure, and restraint stress.

Restraint-Stress-Induced Immunosuppression Is Associated with Reduced Phagocyte Function and Increased Cell Death in Mice
Upon observing relatively high levels of viable bacteria several hours after bacterial injection (Figure 3F) and higher spleen infiltration of macrophages (Figure 4C), we postulated that the impaired phagocyte function of splenic macrophages, specifically their ability to engage and engulf bacteria, might be responsible for restraint-stress-induced immunosuppression.Using described methods [19,71] to assess the phagocyte function of splenic macrophages, we employed fluorescent nanoparticles (NPs) as phagocyte targets.Intriguingly, we discovered that treatments involving IVIg, cold exposure, and restraint stress all resulted in a suppression of spleen phagocyte engagement with fluorescent NPs (Figure 5).Subsequently, to explore the suppressive effect on the live phagocyte population, we examined NP engagements with live splenocytes and observed that restraint stress also suppressed the NP-engaging ability of live splenocytes (Figure S1).These findings strongly suggest that the suppression of phagocyte function contributes to the immunosuppressive effects induced by IVIg, cold exposure, and restraint stress.
The induction of cell death in leukocytes has been recognized as a regulatory process associated with immunosuppression [72][73][74].Additional cell death analyses were therefore conducted and further revealed that restraint stress but not cold exposure induced higher levels of cell death in populations of total splenocytes (Figure 6A, experiment outline, Figure 6B) and F4/80 + spleen macrophage (Figure 6C).These results clarify that macrophage cell death is involved in restraint-stress-induced immunosuppressive effects.The induction of cell death in leukocytes has been recognized as a regulatory process associated with immunosuppression [72][73][74].Additional cell death analyses were therefore conducted and further revealed that restraint stress but not cold exposure induced higher levels of cell death in populations of total splenocytes (Figure 6A, experiment outline, Figure 6B) and F4/80 + spleen macrophage (Figure 6C).These results clarify that macrophage cell death is involved in restraint-stress-induced immunosuppressive effects.

Restraint-Stress-Induced Pyroptosis of Spleen Macrophage
We observed that restraint stress led to GI injury and infiltration of macrophages in the spleen (Figure 4B,C), indicating the potential involvement of an inflammatory response.Here, we examined the expression of the pro-inflammatory cytokine IL-1β in mouse spleen macrophages and found an increased level of IL-1β expression following restraint stress (Figure 7A, experimental outline; Figure 7B,E, vehicle groups).Since inflammasome component caspase-1 activation can trigger IL-1β maturation and pyroptosis, we also assessed the levels of pyroptosis in F4/80 + spleen macrophages.Our analysis revealed higher levels of pyroptosis (Figure 7C,F, vehicle groups) and spleen infiltration (Figure 7D,G, vehicle groups) in F4/80 + splenic macrophages after restraint stress.To investigate whether the suppression of pyroptosis through treatments with the NLRP3 inflammasome inhibitors OLT1177 and Z-WEHD-FMK could reverse the observed abnormalities induced by restraint stress, we found that OLT1177 and Z-WEHD-FMK treatments both mitigated all the aforementioned pathological responses, including in-  The induction of cell death in leukocytes has been recognized as a regulatory process associated with immunosuppression [72][73][74].Additional cell death analyses were therefore conducted and further revealed that restraint stress but not cold exposure induced higher levels of cell death in populations of total splenocytes (Figure 6A, experiment outline, Figure 6B) and F4/80 + spleen macrophage (Figure 6C).These results clarify that macrophage cell death is involved in restraint-stress-induced immunosuppressive effects.

Restraint-Stress-Induced Pyroptosis of Spleen Macrophage
We observed that restraint stress led to GI injury and infiltration of macrophages in the spleen (Figure 4B,C), indicating the potential involvement of an inflammatory response.Here, we examined the expression of the pro-inflammatory cytokine IL-1β in mouse spleen macrophages and found an increased level of IL-1β expression following restraint stress (Figure 7A, experimental outline; Figure 7B,E, vehicle groups).Since inflammasome component caspase-1 activation can trigger IL-1β maturation and pyroptosis, we also assessed the levels of pyroptosis in F4/80 + spleen macrophages.Our analysis revealed higher levels of pyroptosis (Figure 7C,F, vehicle groups) and spleen infiltration (Figure 7D,G, vehicle groups) in F4/80 + splenic macrophages after restraint stress.To investigate whether the suppression of pyroptosis through treatments with the NLRP3 inflammasome inhibitors OLT1177 and Z-WEHD-FMK could reverse the observed abnormalities induced by restraint stress, we found that OLT1177 and Z-WEHD-FMK treatments both mitigated all the aforementioned pathological responses, including in-

Restraint-Stress-Induced Pyroptosis of Spleen Macrophage
We observed that restraint stress led to GI injury and infiltration of macrophages in the spleen (Figure 4B,C), indicating the potential involvement of an inflammatory response.Here, we examined the expression of the pro-inflammatory cytokine IL-1β in mouse spleen macrophages and found an increased level of IL-1β expression following restraint stress (Figure 7A, experimental outline; Figure 7B,E, vehicle groups).Since inflammasome component caspase-1 activation can trigger IL-1β maturation and pyroptosis, we also assessed the levels of pyroptosis in F4/80 + spleen macrophages.Our analysis revealed higher levels of pyroptosis (Figure 7C,F, vehicle groups) and spleen infiltration (Figure 7D,G, vehicle groups) in F4/80 + splenic macrophages after restraint stress.To investigate whether the suppression of pyroptosis through treatments with the NLRP3 inflammasome inhibitors OLT1177 and Z-WEHD-FMK could reverse the observed abnormalities induced by restraint stress, we found that OLT1177 and Z-WEHD-FMK treatments both mitigated all the aforementioned pathological responses, including increased pyroptosis, elevated pro-inflammatory IL-1β expression, and spleen infiltration (Figure 7B-G, OLT1177 and Z-WEHD-FMK groups vs. vehicle groups).Although Z-WEHD-FMK primarily inhibits caspase 1, with some inhibition of caspase 4 and 5 to a lesser extent, the combined results from OLT1177 and Z-WEHD-FMK treatments lead us to conclude that such rescue effects are mediated through the NLRP3-caspase 1 axis.Because macrophage cell death was not observed in the ambient cold exposure (Figure 6), these results suggest that restraint stress induces NLRP3-inflammasome-mediated macrophage pyroptosis, which is a notable phenomenon associated with the immunosuppressive effects induced by restraint stress.
creased pyroptosis, elevated pro-inflammatory IL-1β expression, and spleen infiltration (Figure 7B-G, OLT1177 and Z-WEHD-FMK groups vs. vehicle groups).Although Z-WEHD-FMK primarily inhibits caspase 1, with some inhibition of caspase 4 and 5 to a lesser extent, the combined results from OLT1177 and Z-WEHD-FMK treatments lead us to conclude that such rescue effects are mediated through the NLRP3-caspase 1 axis.Because macrophage cell death was not observed in the ambient cold exposure (Figure 6), these results suggest that restraint stress induces NLRP3-inflammasome-mediated macrophage pyroptosis, which is a notable phenomenon associated with the immunosuppressive effects induced by restraint stress.

Discussion
The mouse model of restraint stress is a well-established approach utilized to study the physiological, behavioral, and biochemical changes associated with psychological stress in mice [6][7][8].By subjecting mice to restraint and immobilization stress, researchers can uncover pathophysiological alterations linked to anxiety and stressed behavior in experimental animals [6].Our analysis indicates that the application of restraint stress induces anxiety-like behavior in mice (Figure 1), suggesting the involvement of psychological stress in the induction of immunosuppression.

Discussion
The mouse model of restraint stress is a well-established approach utilized to study the physiological, behavioral, and biochemical changes associated with psychological stress in mice [6][7][8].By subjecting mice to restraint and immobilization stress, researchers can uncover pathophysiological alterations linked to anxiety and stressed behavior in experimental animals [6].Our analysis indicates that the application of restraint stress induces anxiety-like behavior in mice (Figure 1), suggesting the involvement of psychological stress in the induction of immunosuppression.
IVIg is a therapeutic preparation of pooled human IgG antibodies derived from thousands of healthy donors and has been widely used in various clinical settings for its immunomodulatory properties [18,19].Apart from its well-established efficacy in treating immune-mediated disorders, accumulating evidence suggests that IVIg can also induce immunosuppressive effects under certain conditions [20][21][22][23][24][25][26][27][28][29].IVIg-induced immunosuppression has garnered considerable interest, given its potential implications in regulating immune responses and controlling autoimmune and inflammatory diseases [20][21][22][23][24][25][26][27][28][29].The underlying mechanisms responsible for this immunomodulatory effect are complex and multifaceted, involving interactions with various immune cell populations and immunoregulatory pathways.While IVIg has shown promise in dampening excessive immune responses, the precise factors that determine its immunosuppressive outcomes remain incompletely understood.Previous findings suggested that IVIg's immunomodulatory properties are dependent on the P-selectin pathway, as evidenced by the lack of rescue effect in ITP observed in P-selectin gene knockout (KO; Selp −/− ) mice [19].Likewise, ambient cold exposure depends on functional P-selectin and circulating Ig expressions to elicit the anti-inflammatory response, as demonstrated by the loss of immunosuppressive effect in Pselectin knockout (Selp −/− ) and B-cell-deficient (Ig heavy constant mu [Ighm] −/− ) mice [18].In this present study, we found that restraint stress induces comparable immunosuppressive effects to IVIg treatments and ambient cold exposure.However, restraint-stress-induced immunosuppression does not operate through a P-selectin-dependent mechanism.
These experimental results support the idea that P-selectin may serve as a drug target to treat IVIg and cold-induced immunosuppression.As the immunosuppressive response relies on P-selectin, the administration of P-selectin antagonists may counteract this effect.This hypothesis is reinforced by the observation that immunosuppression is eliminated in mice lacking P-selectin [18,19].On the other hand, restraint-stress-induced immunosuppression does not rely on the P-selectin pathway.Therefore, using P-selectin antagonists would not be effective in suppressing this type of immunosuppression.However, it is worth noting that P-selectin does play a critical role in protecting against restraint-stressinduced GI leakage, as demonstrated in previous studies [15].This suggests that although P-selectin is not involved in the induction of immunosuppression, it still plays a significant role in preventing injury associated with restraint stress.
The reasons behind the significant level of immunosuppression induced by restraint stress and the different pathways through which various stressors, such as ambient cold exposure and restraint stress, induce immunosuppression remain unclear.Through the use of an experimental model involving the administration of Evans blue dye to restrained mice [14][15][16][17], we have demonstrated that restraint stress causes gut leakage in mice (Figure 4A, experiment outline, Figure 4B).Since the spleen serves as a key lymphoid organ responsible for filtering blood, including blood from the GI system, to remove pathogens and foreign substances, it is reasonable to observe leukocyte infiltration into the spleen of mice following restraint stress (Figures 4C and 7D).To restore the homeostasis of the immune system, stress hormones may be elicited as a response to restraint stress, leading to the induction of leukocyte cell death.However, the specific underlying mechanisms involved in this process remain largely unknown and warrant further investigation.
Compared to ambient cold exposure, spleen macrophage cell death is a significant observation in this context.Restraint stress induces GI leakage and injuries, leading to proinflammatory responses such as IL-1β expression and pyroptosis.The invasion of bacterial components, such as lipopolysaccharide (LPS), due to GI leakage requires macrophages to engulf these compounds to maintain GI homeostasis.Toll-like receptor 4, known as the LPS receptor, has been shown to mediate chronic restraint-stress-induced immune suppression [75], suggesting a potential mechanism that may also apply to acute stress.The role of pyroptosis in restraint-stress-induced immunosuppression is not extensively discussed.A report has indicated that caspase-1 inhibitor VX765 suppresses the production of inflammatory factors, reduces the inflammatory response, and protects the gastric mucosa of mice with acute gastric ulcers induced by cold-restraint stress [76].This finding is somewhat in agreement with our results, while the detailed mechanism that leads to immunosuppression in acute stress remains elusive.
At the same time, in the context of anti-cancer immune responses, more evidence suggests that pyroptosis plays a role in generating an immunosuppressive effect.For example, it has been shown that pyroptosis and cytokine secretion can promote cancer progression by evading immune surveillance [72].Helicobacter pylori was found to induce pyroptosis, thereby reducing host anti-tumor immunity [73].Another study highlighted that NLRP3-induced pyroptosis and IL-1β secretion create an immune suppressive environment in breast cancer [74].These studies shed light on the role of pyroptosis triggered by inflammasomes, which can regulate immunosuppression or immune responses within the tumor microenvironment.Given the elusive nature of the role and detailed mechanism of macrophage pyroptosis in restraint-stress-induced immunosuppression, these findings may provide valuable insights for future studies.
Although valuable results have been obtained in this study, it also unveils several limitations that merit additional attention.For instance, the study effectively demonstrated the impact of restraint stress on anxiety-like behaviors in C57Bl/6J mice.However, the extrapolation of these findings to immune disturbances induced by psychological stress in humans necessitates further investigation.Furthermore, the mechanism underlying the immunosuppression induced by restraint stress diverges from that triggered by IVIg and ambient cold exposure.Nevertheless, this mechanism remains enigmatic and calls for further exploration.Notably, the application of NLRP3 inflammasome inhibitors to suppress pyroptosis and mitigate stress-induced effects unveiled the potential involvement of macrophage pyroptosis in restraint-stress-induced immunosuppression.However, the precise utilization of these inhibitors to counteract stress-induced immunological disruptions remains unclear and warrants deeper investigation.Overall, while the paper adeptly delves into the intricacies of restraint-stress-induced immunosuppression, a more thorough examination of the underlying mechanisms and their broader physiological implications might be necessary to foster a more comprehensive comprehension of the observed phenomenon.
In summary, findings in this report indicate that the mechanism of immunosuppression differs between restraint stress, IVIg, and ambient cold exposure in mice.While IVIg and cold exposure induce immunosuppression through a pathway involving P-selectin, restraint stress exerts immunosuppressive effects through a different pathway independent of P-selectin.The experiments show that three treatments (IVIg, cold exposure, and restraint stress) differentially ameliorate ITP and exacerbate suppressed bacterial clearance in P-selectin-deficient (Selp −/− ) mice, further supporting the involvement of different pathways.Additionally, restraint stress reduces phagocyte function, evidenced by suppressed engagement between splenic macrophages and fluorescent nanoparticles, and leads to increased cell death in total splenocytes and F4/80 + splenic macrophages.Furthermore, restraint stress, but not ambient cold exposure, leads to pyroptosis of spleen macrophages, suggesting the potential involvement of macrophage cell death in restraint-stress-induced immunosuppression.Notably, treatments with NLRP3 inhibitors OLT1177 and Z-WEHD-FMK effectively rescue stress-induced macrophage pyroptosis, pro-inflammatory cytokine IL-1β expression, and spleen infiltration.The findings presented here carry important implications for the advancement of treatments aimed at addressing stress-induced immunosuppression.They underscore the necessity for deeper exploration of the underlying mechanisms involved.

Open Field Test (OFT) Behavior Test
Using a modified method [50], after 20 h of restraint stress and a subsequent 2 h period of food and water resupply, an OFT behavior test was employed to measure anxiety-, and depression-like behaviors in mice.The test involved using an open-field device, which consisted of a square box measuring 20 cm × 20 cm with surrounding walls that were 30 cm in height.The central area of the arena, measuring 10 cm × 10 cm, was designated as the center zone.Video recordings were captured using an iPhone Xs Max and later analyzed using ToxTrac software (version 2.98).The distance between the mice and the camera during recording was maintained at 60 cm.

Restraint Stress and Restraint-Stress-Induced GI Leakage
An Evans-blue-fed restraint-stress mouse model was employed as described [14][15][16][17].Mice of ages 10-15 weeks and body weights greater than 26 g were placed in a 50 mL plastic falcon tube for 9 h to induce restraint stress [14,80,81].To ensure adequate air supply, holes were created at the tapering end of the falcon tube.Blood samples of 50 µL were collected at 0, 5, and 9 h after the stress challenge.Acute restraint stress involved subjecting the mice to a single 5-20 h stress session.In the 5-9 h courses, the experiments were performed during the night (10:00 p.m.-7:00 a.m.), predominantly within their dark cycle or active phase, to minimize the influence of circadian rhythm on the results.Both the no stress and restraint stress groups were deprived of food and water during the restraint stress period.Immediately before the stress challenge began, the mice were administered Evans blue (1.2 g/kg, Santa Cruz Biotechnology, Santa Cruz, CA, USA) via a steel feeding tube [14][15][16][17].Blood plasma was obtained by collecting blood in an Eppendorf tube and mixing it with an equal amount of anti-coagulant citrate dextrose solution to prevent coagulation [41][42][43].The collected plasma was then transferred to 96-well plates, and the concentration of Evans blue was measured at 620 nm using a full-spectrum analyzer (Multiskan Spectrum, Thermo Fisher Scientific, Waltham, MA, USA).

Analysis of Bacterial Clearance
The bacterium E. coli was cultured using standard methods [85][86][87].To analyze the immunosuppressive effects of the treatments of IVIg, cold exposure, and restraint stress on bacterial clearance, the experimental mice were challenged by intravenously administering bacteria (E.coli, BL21, 6 × 10 9 CFU/kg; no mortality within 24 h at 4 • C); a 40 h circulating equilibrium at a 25 • C environment was required before treatments of IVIg, cold exposure (control groups: 25 • C exposure), or restraint stress.One day after treatments with IVIg, cold exposure, and restraint stress the spleens of the euthanized mice were collected and weighed.After homogenization (homogenizer, BioSpec Products, Racine, WI, USA) in PBS, surviving bacteria (colony forming units [CFUs]) in the spleen tissues were quantified using the standard plating method.

Measurements of Corticosterone and Cortisol
The plasma levels of corticosterone and cortisol were measured using ELISA (corticosterone and cortisol kits: Cayman Chemical).

Statistical Analyses
The experimental results were analyzed using Microsoft Office Excel version 2003 and SPSS 17 and reported as mean ± standard deviation.The statistical significance of the obtained results was examined using one-way analysis of variance and post-hoc Bonferronicorrected t test.A probability of type 1 error α = 0.05 was considered the threshold of statistical significance.

Conclusions
Our results revealed that restraint stress in C57Bl/6J mice led to anxiety-like behaviors, indicating the involvement of psychological stress.The immunosuppression caused by restraint stress was distinct from that of IVIg and cold exposure, involving reduced phagocyte function and increased spleen macrophage pyroptosis.Treatments with NLRP3 inflammasome inhibitors OLT1177 and Z-WEHD effectively reversed the immunosuppressive effects induced by restraint stress.These findings suggest that macrophage pyroptosis could be a potential therapeutic target for mitigating the impact of stress-induced immunosuppression.

Figure 1 .
Figure 1.Restraint stress resulted in the development of anxiety-like behaviors in mice.During the 20 h stress procedure, both the no stress and stress groups were deprived of access to food and water.After the termination of restraint stress, both the unstressed and stressed groups of mice were given access to food and water for a period of 2 h to restore their resources.Subsequently, the OFT was conducted.Representative video tracking images captured during a 5-min OFT are presented (A).A comparison was made between the control group (no stress) and the restraint stress group (stress) for total distance traveled (B), distance traveled in the central zone (C), and time spent in the central area (D).The number of samples used for analysis was 3 (n = 3).* indicates statistical significance at p < 0.05, ** indicates statistical significance at p < 0.01, when compared to their respective no stress groups.

Figure 1 .
Figure 1.Restraint stress resulted in the development of anxiety-like behaviors in mice.During the 20 h stress procedure, both the no stress and stress groups were deprived of access to food and water.After the termination of restraint stress, both the unstressed and stressed groups of mice were given access to food and water for a period of 2 h to restore their resources.Subsequently, the OFT was conducted.Representative video tracking images captured during a 5-min OFT are presented (A).A comparison was made between the control group (no stress) and the restraint stress group (stress) for total distance traveled (B), distance traveled in the central zone (C), and time spent in the central area (D).The number of samples used for analysis was 3 (n = 3).* indicates statistical significance at p < 0.05, ** indicates statistical significance at p < 0.01, when compared to their respective no stress groups.

Figure 4 .
Figure 4. Time-dependent increase in GI leakage, splenic macrophage infiltration, and stress hormone levels in response to the stress in experimental mice.(A,D) Experimental outlines.(B) Plasma levels of Evans blue.(C) Levels of F4/80 + macrophages in the spleen.(E) Plasma corticosterone levels.(F) Plasma cortisol levels.* p < 0.05, compared to respective 0 h untreated groups; # p < 0.05, compared to respective no stress (−stress) groups.The study involved six mice per group, with three experiments conducted in total (n = 6).

Figure 4 .
Figure 4. Time-dependent increase in GI leakage, splenic macrophage infiltration, and stress hormone levels in response to the stress in experimental mice.(A,D) Experimental outlines.(B) Plasma levels of Evans blue.(C) Levels of F4/80 + macrophages in the spleen.(E) Plasma corticosterone levels.(F) Plasma cortisol levels.* p < 0.05, compared to respective 0 h untreated groups; # p < 0.05, compared to respective no stress (−stress) groups.The study involved six mice per group, with three experiments conducted in total (n = 6).

Figure 6 .
Figure 6.Treatments of IVIg, cold exposure, and restraint stress on the induction of cell death in the population of total splenocytes and splenic macrophages.(A) Experiment outline.(B) Percentage cell death of total splenocytes.(C) Percentage cell death of F4/80 + splenic macrophages.* p < 0.05, vs. respective 0 h groups; # p < 0.05, vs. respective no treatment groups [e.g., 25 °C vs. 4 °C; no stress (−stress) vs. stress].n = 6 (three experiments with total six mice per group).

Figure 6 .
Figure 6.Treatments of IVIg, cold exposure, and restraint stress on the induction of cell death in the population of total splenocytes and splenic macrophages.(A) Experiment outline.(B) Percentage cell death of total splenocytes.(C) Percentage cell death of F4/80 + splenic macrophages.* p < 0.05, vs. respective 0 h groups; # p < 0.05, vs. respective no treatment groups [e.g., 25 °C vs. 4 °C; no stress (−stress) vs. stress].n = 6 (three experiments with total six mice per group).

Figure 6 .
Figure 6.Treatments of IVIg, cold exposure, and restraint stress on the induction of cell death in the population of total splenocytes and splenic macrophages.(A) Experiment outline.(B) Percentage cell death of total splenocytes.(C) Percentage cell death of F4/80 + splenic macrophages.* p < 0.05, vs. respective 0 h groups; # p < 0.05, vs. respective no treatment groups [e.g., 25 • C vs. 4 • C; no stress (−stress) vs. stress].n = 6 (three experiments with total six mice per group).